Molecular Mechanism Of Capsella Bursa-pastoris Resistant To Tribenuron-methyl In Wheat Fields

Wheat is one of the major crops in China and planted area is only less than rice. Shepherd’s purse （Capsella bursa-pastoris （L.） Medik.） is one of important broadleaf weeds that affect the wheat yield in wheat fields and tribenuron is a common sulfonylurea herbicide to controle the broadleaf weeds in wheat fields. After used for15years, tribenuron is incapable of controlling C. bursa-pastoris effectively at recommended use rate (11.25g a.i. ha^-1). In order to elucidate the molecular basis for resistance to tribenuron in C. bursa-pastoris, the generations of the seed collected from Zhumadian in He’nan province is used as experimental material to detected the resistant level with whole-plant bioassays. According to the result of whole-plant bioassays, the generations （Cx-Ri is resistant to tribenuron and Cx-S₁is susceptible to tribenuron） of the seeds collected from Changxing town is used as experimental material for follow-up tests including study on ALS activity in vitro, mutation of ALS gene and expression of ALS gene between resistant and susceptible populations to characterize the molecular basis for resistance to tribenuron in C. bursa-pastoris. Main results as follows:（1） The results of whole-plant bioassays:the generations of the seeds collected from Zhumadian in Henan province were highly resistant to tribenuron. The resistant population Cx-R₁that is the genertation of the seeds collected from Changxing town is highly resistant to tribenuron with the resistance index (GR₅₀ Cx-R₁)/(GR₅₀ Cx-S₁) up to359.3. The resistant population Cx-Ri that is the genertation of the seeds collected from Liangzhu town was also highly resistant to tribenuron with the resistance index (GR₅₀ Lz-R₁)/(GR₅₀ Lz-S₁) up to236.6.（2） The results of ALS activity in vitro:the ALS enzyme of the resistant population （Cx-R₁） express lower sensitivity to tribenuron than that of the susceptible population （Cx-S₁）. The IC₅₀of the resistant and susceptible populations to tribenuron were0.996and0.055μM respectively and the resistance index was18.1. The reduced sensitivity of ALS was one reason for Cx-Ri population highly resistant to tribenuron.（3） The full length cDNA sequence of ALS gene containes2249bp, which includes an open reading frame （ORF） of1644bp which encode547amino acid residues,406nucleotides of5’untranslated sequence （UTR） and199nucleotides of3’UTR. The accession numbers in GenBank were HQ880661and HQ880660respectively. Analysis of the nucleotide and deduced amino acid sequences between the populations indicated that one substitution had occurred in domain A, cytosine by thymine （CCT to TCT） at position197, that led to a change of the amino acid proline in the susceptible to serine in the Cx-R₁. This point mutation was considered as an important reason of the resistance of ALS in Cx-R₁to tribenuron.（4） Using SYBR Green â…  Real-time PCR test, the expression of ALS gene in Cx-R₁and Cx-S₁was determined after spraying the tribenuron at the dosage of11.25g ai ha^-1. Both Cx-R₁and Cx-S₁populations had the same trend, which was ascending at the beginning and then recovered. The results indicated that the expression of ALS gene in Cx-R₁population increased slightly at2d after treatment （DAT） and reached the peak value at4DAT which was4.69times higher than that of control, then it returned to its control level. Meanwhile, the expression of ALS gene Cx-S₁decreased slightly at2DAT, even though it also reached the peak value at4DAT which was2.96times higher than that of control. So the expression of target gene might be one reason for the high resistance.In conclusion, the Cx-R₁population expressed highly resistant to tribenuron due to the reduced sensitivity of ALS to tribenuron and overexpression of ALS in Cx-R₁population. The changed target-site with the mutation （Pro to Ser） at position197is the reason for the reduced sensitivity of ALS to tribenuron in Cx-R₁population.